[Evaluation in vitro of the cardiotoxic effects of 5-fluorouracil for the prevention of cardiovascular damage in health workers occupationally exposed]. / Valutazione in vitro degli effetti cardiotossici da 5-fluorouracile per la prevenzione dei danni cardiovascolari negli operatori sanitari professionalmente esposti.

The occupational exposure to antineoplastic drugs in health care workers determines a risk of absorption through inhalation of vapors or skin contact with drops. Even if many data confirm the cardiotoxicity of anthracyclines, is not clear the evidence of cytotoxicity of 5-Fluorouracil, thoug in a percent of patients receiving this chemotherapy, there is the presence of heart pain, aspecific ECG disorders and induction of coronary disease. This experimental study wants to analyze on the H9c2 cardiomyocyte cell model the effects of 5-Fluorouracil, commonly used in hospital realities of the South Italy, for the prevention of the possible cardiovascular damage in workers occupationally exposed.

Hominoid-specific SPANXA/D genes demonstrate differential expression in individuals and protein localization to a distinct nuclear envelope domain during spermatid morphogenesis.

Human sperm protein associated with the nucleus on the X chromosome consists of a five-member gene family (SPANXA1, SPANXA2, SPANXB, SPANXC and SPANXD) clustered at Xq27.1. Evolved from an ancestral SPANX-N gene family (at Xq27 and Xp11) present in all primates as well as in rats and mice, the SPANXA/D family is present only in humans, bonobos, chimpanzees and gorillas. Among hominoid-specific genes, the SPANXA/D gene family is considered to be undergoing rapid positive selection in its coding region. In this study, RT-PCR of human testis mRNA from individuals showed that, although all SPANXA/D genes are expressed in humans, differences are evident. In particular, SPANXC is expressed only in a subset of men. The SPANXa/d protein localized to the nuclear envelope of round, condensing and elongating spermatids, specifically to regions that do not underlie the developing acrosome. During spermiogenesis, the SPANXa/d-positive domain migrated into the base of the head as the redundant nuclear envelope that protrudes into the residual cytoplasm. Post-testicular modification of the SPANXa/d proteins was noted, as were PEST (proline, glutamic acid, serine, and threonine rich regions) domains. It is concluded that the duplication of the SPANX-N gene family that occurred 6-11 MYA resulted in a new gene family, SPANXA/D, that plays a role during spermiogenesis. The SPANXa/d gene products are among the few examples of X-linked nuclear proteins expressed following meiosis. Their localization to non-acrosomal domains of the nuclear envelope adjacent to regions of euchromatin and their redistribution to the redundant nuclear envelope during spermiogenesis provide a biomarker for the redundant nuclear envelope of spermatids and spermatozoa.

Effect of Staphylococcus enterotoxin B on the concurrent CD8(+) T cell response to influenza virus infection.

Bacterial superantigens have potent in vivo effects. Respiratory viral infections are often associated with secondary bacterial infections, raising the likelihood of exposure to bacterial superantigens after the initiation of the anti-viral immune response. In this study, the general and V beta-specific effects of exposure to Staphylococcal enterotoxin B (SEB) during influenza virus infection on both the ongoing acute and the subsequent recall CD8(+) T cell responses were analyzed, using the well-characterized murine influenza model system and tetrameric MHC/peptide reagents to directly identify virus-specific T cells. The results show that although superantigen exposure during the primary viral infection caused delayed viral clearance, there was remarkably little effect of SEB on the magnitude or TCR repertoire of the ongoing cytolytic T cell response or on the recall response elicited by secondary viral infection. Thus, despite the well-characterized immunomodulatory effects of SEB, there was surprisingly little interference with concurrent anti-viral immunity.

Apparent MHC-independent stimulation of CD8+ T cells in vivo during latent murine gammaherpesvirus infection.

Like EBV-infected humans with infectious mononucleosis, mice infected with the rodent gammaherpesvirus MHV-68 develop a profound increase in the number of CD8+ T cells in the circulation. In the mouse model, this lymphocytosis consists of highly activated CD8+ T cells strikingly biased toward V beta 4 TCR expression. Moreover, this expansion of V beta 4+CD8+ T cells does not depend on the MHC haplotype of the infected animal. Using a panel of lacZ-inducible T cell hybridomas, we have detected V beta 4-specific T cell stimulatory activity in the spleens of MHV-68-infected mice. We show that the appearance and quantity of this activity correlate with the establishment and magnitude of latent viral infection. Furthermore, on the basis of Ab blocking studies as well as experiments with MHC class II, beta2-microglobulin (beta2m) and TAP1 knockout mice, the V beta 4-specific T cell stimulatory activity does not appear to depend on conventional presentation by classical MHC class I or class II molecules. Taken together, the data indicate that during latent infection, MHV-68 may express a T cell ligand that differs fundamentally from both conventional peptide Ags and classical viral superantigens.

Murine gammaherpesvirus M2 gene is latency-associated and its protein a target for CD8(+) T lymphocytes.

Murine gammaherpesvirus 68 (MHV-68) infection of mice is a potential model with which to address fundamental aspects of the pathobiology and host control of gammaherpesvirus latency. Control of MHV-68 infection, like that of Epstein-Barr virus, is strongly dependent on the cellular immune system. However, the molecular biology of MHV-68 latency is largely undefined. A screen of the MHV-68 genome for potential latency-associated mRNAs revealed that the region encompassing and flanking the genomic terminal repeats is transcriptionally active in the latently infected murine B-cell tumor line S11. Transcription of one MHV-68 gene, that encoding the hypothetical M2 protein, was detected in virtually all latently infected S11 cells and in splenocytes of latently infected mice, but not in lytically infected fibroblasts. Furthermore, an epitope was identified in the predicted M2 protein that is recognized by CD8(+) T cells from infected mice and a cytotoxic T lymphocyte line that recognizes this epitope killed S11 cells, indicating that the M2 protein is expressed during latent infection and is a target for the host cytotoxic T lymphocyte response. This work therefore provides essential information for modeling MHV-68 latency and strategies of immunotherapy against gammaherpesvirus-related diseases in a highly tractable animal model.

Bacterial superantigens reactivate antigen-specific CD8+ memory T cells.

Superantigens stimulate naive CD4+ and CD8+ T cells in a TCR V beta-specific manner. However, it has been reported that memory T cells are unresponsive to superantigen stimulation. In this study, we show that staphylococcal enterotoxins (SE) can activate influenza virus-specific CD8+ memory cytotoxic T cells. In vivo SEB challenge of mice that had recovered from influenza virus infection (memory mice) resulted in the generation of vigorous influenza-specific cytotoxic T lymphocyte (CTL) activity and in vitro SEA or SEB stimulation of splenic T cells from memory mice, but not naive mice, also induced influenza-specific CTL. Analysis of the mechanism of activation suggested that although there may be a component of cytokine-mediated bystander activation, the CTL activity is largely generated in response to direct TCR engagement by superantigen. Moreover, influenza-specific CTL could be generated from purified CD8+ CD62L loCD44hi (memory phenotype) T cells cultured in the presence of T cell-depleted splenic antigen-presenting cells and SE. Purified CD8+ memory T cells also secreted lymphokines and synthesized DNA in response to superantigen. These results definitively demonstrate that CD8+ memory T cells respond to SE stimulation by proliferating and developing appropriate effector function. Furthermore, the data raise the possibility that otherwise inconsequential exposure to bacterial superantigens may perturb the CD8+ T cell memory pool.

Efficient priming of CD8+ memory T cells specific for a subdominant epitope following Sendai virus infection.

The relationship between the primary effector CTL response to viral infection and the subsequent pool of memory CTL precursors (CTLp) is poorly understood. Here, we have analyzed the induction of both effector CTL and memory CTLp to dominant and subdominant epitopes following Sendai virus infection of C57BL/6 mice. A single peptide derived from the Sendai virus nucleoprotein (NP(324-332)) binds to both H-2 Kb and Db MHC class I molecules, generating both immunodominant (NP(324-332)/Kb) and subdominant (NP(324-332)/Db) epitopes. Following intranasal Sendai virus infection, NP(324-332)/Kb-specific CTL dominated the primary effector CTL response in the lung and were present at high frequency in the memory CTLp pool. In contrast, NP(324-332)/Db-specific CTL were not a detectable component of the effector response to primary Sendai virus infection. However, memory CTLp specific for this subdominant epitope were induced at frequencies approaching those of CTLp specific for the immunodominant epitope. These data indicate that memory CTLp specific for subdominant epitopes can be primed by Sendai virus infection in the absence of a detectable effector response. To determine whether CTLp memory to subdominant epitopes is functional in the context of Sendai virus infection, memory CTLp specific for a subdominant epitope were selectively primed by vaccination. These cells dominated the subsequent effector CTL response to Sendai virus infection, demonstrating that memory CTLp primed against subdominant epitopes can participate in an immune response and effectively compete with T cells specific for immunodominant epitopes. These data have implications for the development of vaccines designed to emphasize cellular immunity.

In vivo effects of locally secreted IL-10 on the murine antitumor immune response.

BACKGROUND: Interleukin-10 (IL-10) is a cytokine secreted by the TH2 class of murine lymphocytes that suppresses the secretion of interferon-gamma (IFN-gamma) by TH1 lymphocytes and inhibits macrophage-mediated T-cell stimulation and cytotoxicity. The observation that IL-10 is produced by human carcinomas in vitro and in vivo led to the hypothesis that this cytokine plays a role in the suppression of the human anti-tumor immune response. We tested this hypothesis in a murine model. METHODS: To evaluate the effect of IL-10 on the induction of an anti-tumor immune response, mice were immunized with tumor cells transfected with the IL-10 gene and then challenged with parental tumor. The effect of the local secretion of IL-10 on an established immune response was tested by immunizing mice with parental tumor and then challenging with IL-10-secreting tumors. RESULTS: IL-10-secreting tumors were as effective immunogens as control tumors. Immune mice rejected IL-10-secreting tumors as readily as control challenge tumors. In an in vitro assay, IL-10 did not inhibit CD8 lymphocyte secretion of IFN-gamma in response to tumor stimulation. One IL-10-secreting tumor clone regressed when injected into naive mice and induced an antigen-specific immune response capable of protecting mice from subsequent tumor challenge. CONCLUSIONS: The local secretion of IL-10 did not inhibit either the induction of an antitumor immune response or the ability of established effector cells to reject challenge tumors. In contrast to its effect on TH1 lymphocytes, IL-10 does not inhibit IFN-gamma secretion by CD8 lymphocytes.

Impaired generation of anti-AKR/Gross murine leukemia virus cytotoxic T lymphocytes in mice experimentally infected with MuLV.

C57BL/6 (B6) and C57BL/6.Fv-1n (B6.Fv-1n) mice mount AKR/Gross murine leukemia virus (MuLV)-specific cytolytic T lymphocyte (CTL) responses following primary and secondary stimulation with AKR/Gross MuLV-induced tumor cells. In contrast, mice exposed to infectious virus rather than virus-infected cells generate little, if any, antiviral CTL activity. In this report, we show that inoculation of B6 or B6.Fv-1n mice with MuLV prior to priming with H-2-matched AKR/Gross virus antigen-positive tumor cells resulted in a profound inhibition of the virus-specific CTL response. Antiallogeneic major and minor histocompatibility antigen-specific CTL responses were not significantly diminished in MuLV-infected mice. The AKR/Gross MuLV-specific CTL response in B6 mice was inhibited by NB-tropic (SL3-3NB, Friend and Moloney), but not N-tropic (AKR623) MuLV, suggesting that productive infection of host cells was required. We were unable to inhibit the in vitro generation of virus-specific CTL by adding modulator cells from virus-infected mice to mixed lymphocyte-tumor cell cultures (MLTC) of spleen cells from uninfected animals. We also failed to augment CTL generation in MLTC from virus-infected animals by adding exogenous IL-2 or CD4+ lymphocytes from uninfected, tumor-primed mice. Taken together, the data suggested that the inhibition resulted from either a direct or an indirect effect on the in vivo priming of virus-specific CD8+ cells. It is therefore interesting that MuLV such as Friend and Moloney, which do not encode the immunodominant epitope recognized by anti-AKR/Gross MuLV CTL, are nonetheless able to specifically inhibit this response. These results demonstrate a potentially important mechanism by which retroviruses may escape CTL-mediated immunity.

Recognition of endogenous ecotropic murine leukaemia viruses by anti-AKR/Gross virus cytotoxic T lymphocytes (CTL): epitope variation in a CTL-resistant virus.

AKR/Gross virus-specific cytotoxic T lymphocytes (CTL) from C57BL/6 (B6) mice are H-2Kb-restricted and recognize epitopes encoded by the prototype endogenous ecotropic murine leukaemia virus (Emv) AKR623. Four CTL epitopes have been identified by the use of synthetic peptides corresponding to AKR623-encoded amino acid sequences. Here we present both functional and nucleotide sequence data indicating that three closely related Emv share all of these CTL epitopes. We also found that one other murine leukaemia virus (MuLV) was not susceptible to lysis by these CTL. This is the ecotropic component of the LP-BM5 virus complex that causes murine AIDS. Nucleotide sequencing revealed that three of the four epitopes, including the immunodominant peptide, are altered in this virus. The other epitope was unchanged. These data implied that the inability of anti-AKR/Gross virus CTL to lyse cells infected with the LP-BM5 ecotropic (BM5eco) MuLV was due to the functional loss of three of the four CTL epitopes. Using recombinant vaccinia and Sindbis virus vectors, we have shown that the BM5eco-encoded form of the immunodominant epitope, which differs only by an arginine for lysine substitution at the N-terminal residue, fails to induce a CTL response in B6 mice. Immunization with BM5eco-infected cells also failed to induce MuLV-specific CTL. In light of the long in vivo passage history of the LP-BM5 complex in B6 mice, our results are consistent with a contribution of CTL-mediated immune selection to the evolution of the BM5eco MuLV.

Cytotoxic T lymphocyte responses to the envelope proteins of endogenous ecotropic and mink cytopathic focus-forming murine leukemia viruses in H-2b mice.

To study the possible role of immune selection in the in vivo generation of pathogenic recombinant murine leukemia viruses (MuLV), we have constructed recombinant vaccinia viruses (rVV) expressing the envelope genes of three MuLV: AKR623, MCF247, and MCF13. rVV expressing either AKR623 or MCF247 env could prime H-2b mice for anti-AKR/Gross virus CTL responses, and stimulate the in vitro generation of CTL from the spleens of mice immunized with an AKR/Gross virus-positive lymphoma. MC57 (H-2b) cells infected with either 623EnvVac or 247EnvVac could serve as targets for ARK/Gross virus-specific CTL. Cells infected with the rVV expressing MCF13 env, however, were lysed much less efficiently by these CTL. 13EnvVac was also ineffective in priming or stimulating retrovirus-specific CTL. Finally, experiments with synthetic peptides and minigenes suggested that the reduced immunogenicity of the MCF13 envelope protein likely resulted from a single amino acid substitution within an immunodominant epitope of the p15E (TM) protein. The region of MCF13 env that encodes this epitope is derived from an endogenous xenotropic virus, while the allelic sequences in MCF247 are of ecotropic virus origin. These results suggest the potential for recombination within the MuLV envelope gene to allow escape from host cellular immune responses.

An increase in disease latency is associated with a host-dependent selection for recombinant murine leukemia viruses with substitutions in the p15E (TM) gene.

The genomes of recombinant murine leukemia viruses recovered from HRS/J (type I env recombinants) and CWD (type II env recombinants) mice have distinct envelope gene structures. To better understand the biologic significance of these differences, we examined the differences in the responses of HRS/J and CWD mice to inoculation with an oncogenic type II env recombinant. The CWD recombinant accelerated the onset of lymphoma in both strains, but the disease latency in the HRS/J mice was about 2 months longer. Analysis of the recombinant viruses in the HRS/J tumors revealed that the injected type II env recombinant had recombined in vivo with the endogenous ecotropic viruses to generate secondary recombinants with type I envelope genes. In another set of experiments, comparison of complete or partial DNA sequences of the envelope genes from six recombinant proviruses confirmed that the origins of the sequences that encode an amino-terminal region of the TM envelope protein, p15E, distinguish type I envelope genes from type II. Taken together with the results of previous studies, these observations suggest that the differences in the responses of HRS/J and CWD mice to the oncogenic type II env recombinant resulted from an interaction between the viral TM protein and a host factor expressed in HRS/J mice.

Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1-related xenotropic viruses from CWD mice.

The acquisition of U3 region sequences derived from the endogenous xenotropic provirus Bxv-1 appears to be an important step in the generation of leukemogenic recombinant viruses in AKR, HRS, C58, and some CWD mice. We report here that each of three CWD lymphomas produced infectious xenotropic murine leukemia virus related to Bxv-1. In Southern blot experiments, these proviruses hybridized to probes that were specific for the xenotropic envelope and Bxv-1 U3 region sequences. Nucleotide sequence analysis of a cloned CWD xenotropic provirus, CWM-S-5X, revealed that the envelope gene was closely related to but distinct from those of other known xenotropic viruses. In addition, the U3 region of CWM-S-5X contained a viral enhancer sequence that was identical to that found in MCF 247, a recombinant AKR virus that is thought to contain the Bxv-1 enhancer. Finally, restriction enzyme sites in the CWM-S-5X provirus were analogous to those reported within Bxv-1. These results establish that the virus progeny of Bxv-1 have the potential to donate pathogenic enhancer sequences to recombinant polytropic murine leukemia viruses. Interestingly, the three CWD polytropic viruses that were isolated from the same tumor cells that produced the Bxv-1-like viruses had not incorporated Bxv-1 sequences into the U3 region.

Oncogenicity and U3 region sequences of class II recombinant MuLVs of CWD mice.

The pathogenic potential of Class II env recombinant murine leukemia viruses (MuLV) found in the high leukemia strain CWD has not been defined. We found that neonatal CWD mice that were injected with the phenotypic mixture of the spontaneous CWD class II env recombinant, CWM-T-15, and the AKR endogenous ecotropic virus, Akv 623, developed non-T-cell lymphomas more rapidly than controls inoculated with either virus alone or with a CWD ecotropic virus. In contrast, CWN-T-25, a class II env MuLV that was recovered from a CWD mouse injected with the AKR ecotropic virus SL3-3, dramatically accelerated the onset of T-cell lymphomas in the same assay. Southern blots of the tumor DNAs from each set of animals revealed the integration of recombinant and ecotropic proviruses. We also found that there were differences in the nucleotide sequences of the viral enhancer elements of the CWD viruses. The results indicate that (1) the two CWD class II env recombinants that were tested contained oncogenic determinants; (2) phenotypic mixing with ecotropic viruses was required for the full expression of the pathogenic potential of the CWM-T-15 recombinant; and (3) the distinct phenotypes of the CWD viruses likely reflected the differences in the origin of the viral enhancer element.

A host gene regulates the structure of the transmembrane envelope protein of murine leukemia viruses.

Heterogeneity in the structure of the envelope proteins has been observed in many human and animal retroviruses and may influence pathogenicity. However, the biological significance of this heterogeneity and the mechanisms by which it is generated are poorly understood. We have studied a mouse model in which the envelope gene structure of lymphoma-associated viruses appears to be controlled by a single host gene. The inoculation of HRS and CWD mice with a leukemogenic murine leukemia virus (MuLV) results in recombination between the injected virus and envelope gene sequences of endogenous retroviruses. The genomes of HRS (class I) env recombinants and CWD (class II) env recombinants differ in the sequences encoding the NH2-terminal portion of the transmembrane envelope protein (TM). We have shown that an HRS gene linked to the MHC on chromosome 17 mediates a dominant selection for recombinant retroviruses with the class I envelope gene structure. CBA mice, which share the H-2k haplotype with HRS, also carry the dominant allele at this locus. This system provides a useful model for studies of host factors involved in the selection of specific variants of pathogenic retroviruses.

Association of recombinant murine leukemia viruses of the class II genotype with spontaneous lymphomas in CWD mice.

We determined the phenotype and genotype of murine leukemia viruses associated with the development of spontaneous nonthymic lymphomas in the high-leukemia mouse strain CWD/J. By T1 oligonucleotide fingerprint analysis of the viral RNA, the ecotropic viruses recovered from the spleen or thymus of preleukemic CWD/J mice were found to represent the progeny of the two endogenous ecotropic proviruses present in this strain. Polytropic murine leukemia viruses were produced by tissues from one-half of the leukemic mice, and fresh tumor cells from one of the two animals tested expressed recombinant envelope glycoproteins. The genomic structure of the recombinant viruses resembled those of class II polytropic viruses of NFS X Akv mice and differed from those of class I recombinant viruses that are commonly isolated from other high-leukemia strains such as AKR and HRS. Acquired retroviral sequences with the structural features of class II recombinant proviruses were detected in the DNA from each CWD/J tumor by the Southern blot technique. Finally, the injection of a mixture of CWD/J ecotropic and class II recombinant polytropic viruses into neonatal CWD/J mice accelerated the onset of lymphoma, whereas the endogenous ecotropic virus was inactive in these assays.

Lithium stimulation of murine hematopoiesis in liquid culture: an effect mediated by marrow stromal cells.

Lithium has previously been observed to stimulate in vitro Dexter culture hemopoiesis with increases in granulocytes, megakaryocytes, and pluripotent stem cells (CFU-S). In the present study, a two-phase murine Dexter culture system was established to study the mechanism of lithium-mediated stem cell stimulation. Different lots of horse sera or fetal calf sera were found to have markedly different effects on Dexter culture growth; given the appropriate sera supplementation, supernatant cells from Dexter cultures established from C57BL/6J mice 3 wk previously were free of stromal-forming capacity, but had stem cells and could grow on 900-950 R irradiated stroma. Conversely, in vitro irradiation (900-950 R) of 3-wk cultures resulted in a stem-cell-free adherent monolayer that could support growth for up to 9 wk in culture. The stroma from Dexter cultures preexposed to lithium chloride (1.0 mmole/liter) for 3 wk, irradiated (900 R), and then refed with 3-wk Dexter supernatant cells has an enhanced capacity to support cell production, CFU-S, and probably granulocyte-macrophage colony-forming cell (GM-CFU-C) production, as compared to stroma not preexposed to lithium. Lithium carryover was ruled out in these experiments. These data indicate that lithium stimulates CFU-S and in vitro granulopoiesis by an indirect effect on a radioresistant adherent stromal cell.

Cryopreservation of marrow and stromal progenitor cells: use of long-term liquid culture as a measure of the recovery of renewable hematopoietic and stromal cells.

Long-term liquid culture was used to assess the broad functional integrity of murine bone marrow after cryopreservation. Cryopreserved and thawed marrow contained not only renewable hematopoietic stem cells, but also stromal progenitor cells capable of transferring the hematopoietic microenvironment to liquid culture. Long-term liquid culture may be a superior method of assessing the functional potential of cryopreserved marrow when compared to stem cell assays alone.